Jaakko Brotherus

Soluble and active renal Na, K-ATPase with maximum protein molecular mass 170,000 +/- 9,000 daltons; formation of larger units by secondary aggregation.

Summary Purified membrane-bound Na,K-ATPase from pig kidney was solubilized with nonionic detergent, dodecyloctaethylenglycol monoether (C 12 E 8 ) as 70–90% active protein units with S 20,w 7.4 ± 0.2 and maximum molecular mass 170,000 ± 9,000 daltons indicating that the soluble complex predominantly consisted of protomeric αβ-units. Inactivation of Na,K-ATPase by excess C 12 E 8 was not related to the aggregation state of the protein. On storage both the soluble Na,K-ATPase and soluble Ca-ATPase (115,000 daltons) from sarcoplasmic reticulum underwent secondary aggregation which may account for previous reports of higher molecular weights.

Soluble and enzymatically stable (Na++K+)-ATPase from mammalian kidney consisting predominantly of protomer αβ-units: Preparation, assay and reconstitution of active Na+, K+transport

Soluble (Na++K+)-ATPase consisting predominantly of αβ-units with Mr below 170 000 was prepared by incubating pure membrane-bound (Na++K+)-ATPase (35–48 μmol Pi/min per mg protein) from the outer renal medulla with the non-ionic detergent dodecyloctaethyleneglycol monoether (C12E8). (Na++K+)-ATPase and potassium phosphatase remained fully active in the detergent solution at C12E8/protein ratios of 2.5–3, at which 50–70% of the membrane protein was solubilized. The soluble protomeric (Na++K+)-ATPase was reconstituted to Na+, K+ pumps in phospholipid vesicles by the freeze-thaw sonication procedure. Protein solubilization was complete at C12E8/protein ratios of 5–6, at the expense of partial inactivation, but (Na++K+)-ATPase and potassium phosphatase could be reactivated after binding of C12E8 to Bio-Beads SM2. At C12E8/protein ratios higher than 6 the activities were irreversibly lost. Inactivation could be explained by delipidation. It was not due to subunit dissociation since only small changes in sedimentation velocities were seen when the C12E8/protein ratio was increased from 2.9 to 46. As determined immediately after solubilization, S20,w was 7.4 S for the fully active (Na++K+)-ATPase, 7.3 S for the partially active particle, and 6.5 S for the inactive particle at high C12E8/protein ratios. The maximum molecular masses determined by analytical ultracentrifugation were 141 000–170 000 dalton for these protein particles. Secondary aggregation occurred during column chromatography, with formation of enzymatically active (αβ)2-dimers or (αβ)3-trimers with S20,w=10–12 S and apparent molecular masses in the range 273 000–386 000 daltons. This may reflect non-specific time-dependent aggregation of the detergent micelles.

Novel stereoconfiguration in lyso-bis-phosphatidic acid of cultured BHK-cells

Abstract Lyso-bis-phosphatidic acid purified from cultured hamster kidney fibroblast cells (BHK-cells) was subjected to strong alkaline hydrolysis. The hydrolysate contained phosphorus, free glycerol, total glycerol, α-glycerophosphate, β-glycerophosphate and sn-glycerol-3-phosphate in mole ratios of 1.0:1.0:1.9:0.4:0.6:0.02. The absence of sn-glycerol-3-phosphate indicates that the backbone of this lipid has the uncommon structure of 1-sn-glycerophosphoryl-1′-sn-glycerol. Consequently, the biosynthesis and the degradation of this lipid must differ from the other known mammalian glycerophospholipids.

The stereochemical configuration of lysobisphosphatidic acid from rat liver, rabbit lung and pig lung

Lysobisphosphatidic acid known also as bis(monoacyl-glycerol)phosphate, was isolated from liver of rats treated with Triton WR1339, and from rabbit and pig lung. Alkaline hydrolysates of all these samples of lysobisphosphatidic acid were essentially similar and contained phosphorus, total glycerol, free glycerol, total glycerophosphates, beta-glycerophosphate, total alpha-glycerophosphates, sn-glycero-1-phosphate and sn-glycero-3-phosphate in a molar ratio of 1.0 : 2.0 : 1.0 : 1.0 :0.6 : 0.4 : 0.38 : 0.04. This proves that the backbone of the principal lysobisphosphatidic acid from all three sources has the structure of 1-sn-glycerophospho-1-sn-glycerol.

Isolation and characterisation of bis-phosphatidic acid and its partially deacylated derivatives from cultured BHK-cells

Abstract Bis-phosphatidic acid, semi-lyso-bis-phosphatidic acid and lyso-bis-phosphatidic acid were isolated from cultured baby hamster kidney fibroblasts (BHK-cells). They constituted about 0.02%, 0.1% and 1.6% respectively of total lipid phosphorus of fresh BHK-cell monolayers. These three phospholipids were also prepared by partial synthesis. The natural lipids were identical with the synthetic samples as determined by thin-layer chromatography and by partial degradation by controlled alkaline methanolysis, acetic acid hydrolysis and acetolysis. The three derivatives of bis-phosphatidic acid contained 60–80% of octadecenoic acid; in this respect they were different from other BHK-cell phospholipids.

Phospholipids of subcellular organelles isolated from cultured BHK cells.

Mitochondrial and nuclei were purified from cultured hamster fibroblasts (BHK21 cells) by centrifugation in sucrose gradients. The phospholipid compositions of the preparations were compared to those of the previously purified plasma membranes, endoplasmic reticulum and lysosomes. The mitochondria had a characteristically high content (approx. 16% of lipid phosphorus) of cardiolipin, which was practically absent from the other purified organelles. The nuclei were enriched in phosphatidylcholine and phosphatidylinositol (approx. 68% and 5% of lipid phosphorus, respectively). Lysobisphosphatidic acid was almost absent from the mitochondria and nuclei, as well as from the plasma membrane and endoplasmic reticulum, which suggests that this phospholipid is confined to the lysosomes of the BHK cell. The nuclei and the mitochondria contained relatively little sphingomyelin, a characteristic lipid of the plasma membrane. The distributions of the total cellular phospholipid and protein between the various organelles were calculated and compared to the corresponding data estimated for the rat liver. The BHK cell contained relatively more phospholipids in the nucleus and the lysosomes than the liver. All the organelles of the BHK cell contained less protein per phospholipid than the equivalent organelles of the liver.

Low and moderate concentrations of lysobisphosphatidic acid in brain and liver of patients affected by some storage diseases.

The relative amount of lysobisphosphatidic acid (LBPA), known also as bis(monoacylglyceryl)phosphate, among the total phospholipids was analyzed in post mortem samples of brain and liver of patients affected by four storage diseases. In spite of the extensive accumulation of storage lysosomes, none of the samples revealed a highly elevated LBPA content comparable to that found in the liver in Niemann-Pick disease and in the liver in lipidosis induced by 4,4′-diethylaminoethoxyhexestrol. We conclude that, although LBPA is often present in high concentration in lysosomes of many types of cells, it is not always a major component of these organelles.

Synthesis of monoacid 2,3-diacyl-sn-glycerols via 1,6-ditrityl-d-mannitol

Abstract Stereochemically pure 2,3-dipalmitoyl- sn -glycerol and 2,3-dioleoyl- sn -glycerol were prepared in an overall yield of 20% by a new and facile method starting from D-mannitol. The synthetic intermediates were 1,6-ditrityl-D-mannitol ( 1 ), 1-trityl- sn -glycerol ( 2 ), and 1-trityl-2,3-diacyl- sn -glycerol ( 3 ). The key reaction was the oxidation of 1 with lead tetraacetate followed by reduction with sodium borohydride. The product ( 2 ) was readily separated from the only byproduct, tritylethyleneglycol.

Phospholipids of outer and inner nuclear membranes in rat liver and BHK-21 cells

Abstract Isolated nuclei of rat liver and BHK-21 cells were treated with citric acid and the resulting outer nuclear membrane sheets were separated from the nuclear residues surrounded by the inner nuclear membrane. Both fractions contained approximately equal amounts of phospholipid in both cell types. The phospholipid compositions of the two fractions were remarkably similar. The results are in accordance with the notion of the structural continuity of the two nuclear membranes.

The active site and the phospholipid activation of rat liver lysosomal lipase are not stereospecific

The stereochemical specificity of lysosomal lipase of rat liver was investigated using enantiomeric triacylglycerol analogs, sn-1-alkyl-2,3-diacylglycerol and sn-3-alkyl-1,2-diacylglycerol as substrates. Lysosomal lipase utilized both substrates with equal rates. The dependence of the activity of lysosomal lipase on the stereoconfiguration of activating acidic phospholipid was also studied. Our results showed that both sn-3-phospholipids (diphosphatidylglycerol, phosphatidylserine) and sn-1-phospholipids (bis(monoacylglycero)phosphate (BMP)) were efficient activators of this enzyme and thus the stereochemical configuration of the activating phospholipid is not important. Accordingly, the rat liver lysosomal lipase lacks stereospecificity with respect to both the triacylglycerol substrate and the acidic phospholipid activator.